Summary: Antibiotic resistance represents one of the greatest threats to human health. In particular, the six so-called ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and enterobacteriaceae) represent highly drug-resistant bacteria that exert a tremendous global burden of disease. The potential scope of this crisis was highlighted in a recent report commissioned by the Wellcome Trust and British Government; the authors projected that, by 2050, drug-resistant bacterial infections could cost the global economy a cumulative $100 trillion and kill 10 million people annually. To address this issue, there is a critical need for innovative antibacterial treatments. One compelling therapeutic strategy leverages recombinant enzymes that degrade cell wall peptidoglycan, thereby causing bacterial lysis and death. Currently, all such lytic enzyme therapies are trans-acting in nature, i.e., they are derived from bacteriophage or the immune systems of eukaryotic organisms. This proposal seeks to establish an entirely new paradigm for developing bacteriolytic enzyme drugs. We hypothesize that a pathogen's own endogenous cell wall hydrolases (i.e., ?autolysins?) can be co-opted to yield potent antimicrobial agents that are refractory to new resistance phenotypes. To test this hypothesis, we will pursue initial studies with the high impact pathogen methicillin resistant S. aureus (MRSA), although the strategy should be broadly applicable to any bacterial pathogen. Here, complementary computational and experimental approaches will be utilized to identify, isolate, and engineer potent autolysins derived from staphylococcal proteomes. In aim 1, the sequenced genome of S. aureus and related bacteria will be searched for autolysins using bioinformatics. Candidate enzymes will be cloned, evaluated, and their activities will be improved via computationally guided fusion to high performance cell wall targeting domains. In aim 2, a complementary high throughput screening strategy will be taken to identify autolysins from genomic libraries of pathogenic staphylococci. The activities of candidate enzymes will be improved via combinatorial chimeragenesis with high performance cell wall targeting domains, followed by high throughput functional screening of the resultant chimeric libraries. In aim 3, lead autolysin candidates will be further engineered for potent anti-staphylococcal activity using a directed evolution strategy. The most promising lead candidates from these studies will be rigorously evaluated using a panel of clinically relevant in vitro and in vivo assays. Ultimately, this project could yield both novel anti-staphylococcal agents and an entirely new paradigm for development of antibacterial biotherapies.